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Cu Transporting ATPase and Diabetic Vascular Complications

铜转运 ATP 酶与糖尿病血管并发症

基本信息

批准号：
9977232
负责人：
TOHRU FUKAI
金额：
$ 68.07万
依托单位：
AUGUSTA UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
2017
资助国家：
美国
起止时间：
2017-09-01 至 2022-07-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/9977232
关键词：
3-Dimensional ATP phosphohydrolase Address Animal Model Animals Binding Binding Sites Bioavailable Biological Assay Biological Availability Biotinylation Blood Vessels Cardiac Cardiovascular system Cells Clinical Complications of Diabetes Mellitus Copper Data Diabetes Mellitus Diabetic Angiopathies Diabetic mouse Disease Down-Regulation Endothelial Cells Endothelium Enzymes Extracellular Space Fluorescence Microscopy Fluorescence Resonance Energy Transfer Functional disorder Gene Transfer Goals Grant Hindlimb Homeostasis Human Hypertension Impairment Inductively Coupled Plasma Mass Spectrometry Inflammation Inflammatory Injections Ischemia Knockout Mice Lead Leukocytes Measures Micronutrients Molecular Morbidity - disease rate Mus Mutate Patients Perfusion Peripheral Peripheral Vascular Diseases Permeability Play Protein-Lysine 6-Oxidase Reporting Risk Factors Roentgen Rays Role Scaffolding Protein Signal Transduction Synchrotrons Testing Tissues Transgenic Mice Transgenic Organisms Ubiquitination Vascular Diseases Vascular Endothelial Growth Factors Vascular Permeabilities angiogenesis base cadherin 5 copper-transporting ATPase critical limb Ischemia diabetic diabetic patient extracellular in vivo innovation insight intravital microscopy live cell imaging migration mortality mutant neovascularization new therapeutic target novel overexpression prevent programs protein protein interaction recruit repaired small hairpin RNA therapeutic target tissue repair transcription factor wound healing

项目摘要

This grant will elucidate the novel protective role of “Copper transporting ATPase (ATP7A)” against impaired reparative neovascularization in diabetic ischemic vascular diseases. Diabetic complication leads to defective neovascularization in ischemic peripheral vascular disease due to impaired angiogenesis and endothelial cell (EC) barrier dysfunction with unknown mechanisms. Copper (Cu), an essential micronutrient, is involved in angiogenesis while excess Cu contributes to inflammatory diseases such as diabetes. Since excess Cu is toxic, bioavailability of intracellular Cu is tightly controlled by ATP7A which delivers Cu to the secretory Cu enzymes, or exports Cu to extracellular space. Our lab discovered that ATP7A in VSMC protects against hypertension. We also identified ‘IQGAP1” as a VEGF receptor2 (VEGFR2) binding scaffold protein promoting VEGF signaling and post-ischemic angiogenesis. However, role of ATP7A in ECs for defective post-ischemic revascularization in diabetes is entirely unknown. Preliminary data suggest that ATP7A prevents VEGFR2 degradation through binding to IQGAP1 and maintains basal EC barrier function via regulating VE-cadherin (VE- Cad). ATP7A expression is markedly decreased in ECs from diabetic mice or microvessels of type2 diabetes patients. ATP7A mutant (ATP7Amut) mice with reduced Cu transport function or diabetic mice show impaired ischemia-induced reparative angiogenesis with excess tissue Cu and vascular permeability/tissue damage, which are rescued by overexpression of ATP7A. We thus hypothesize that ATP7A functions to promote and integrate key vascular repair programs such as angiogenesis and maintaining endothelial barrier function in a Cu-dependent manner, which is required for restoring neovascularization in diabetic ischemic vascular disease. Aim 1 will define the protective role of ATP7A against: i) impaired VEGF-induced angiogenesis by stabilizing VEGFR2 and ii) endothelial barrier dysfunction by maintaining Cu homeostasis in ECs isolated from diabetic mice and human microvessels of type2 diabetic patients. Aim 2 will determine the molecular mechanism by which ATP7A downregulation in diabetes impairs VEGFR2 signaling and endothelial barrier integrity by focusing on; i) ATP7A binding to IQGAP1 that prevents VEGFR2 ubiquitination/degradation in a Cu-independent manner, and ii) role of ATP7A in regulating miR-125b that represses VE-Cad via Cu- dependent transcription factor Atox1. Aim 3 will define the protective role of ATP7A against diabetes-induced impaired post-ischemic neovascularization and tissue repair in vivo and address underlying mechanisms using animal model of critical limb ischemia. We will use ATP7Amut and ATP7A transgenic mice; inducible EC-specific ATP7A-/- or Cu importer CTR1-/- mice or type1 and type2 diabetes mice; innovative ICP-Mass Spec, X-ray fluorescence microscopy to analyze [Cu]i in cells and tissues; FRET or BiFC-based protein-protein interaction; and intravital microscopy. Our study will uncover Cu transporter ATP7A as a novel potential therapeutic target to enhance integrated vascular repair program in patients with diabetic vascular complications.

该基金将阐明“铜转运ATP酶（ATP 7A）”对细胞凋亡的新保护作用。糖尿病缺血性血管疾病中的修复性新生血管形成受损。糖尿病并发症在缺血性外周血管疾病中由于血管生成受损而导致有缺陷的新血管形成，内皮细胞（EC）屏障功能障碍，机制不明。铜（Cu）是一种必需的微量营养素，参与血管生成，而过量的铜会导致糖尿病等炎症性疾病。因为过量铜是有毒的，细胞内铜的生物利用度由ATP 7A严格控制，ATP 7A将铜运送到分泌铜酶，或输出铜到细胞外空间。我们的实验室发现，ATP 7A在VSMC中可以防止高血压我们还鉴定了“IQGAP 1”作为VEGF受体2（VEGFR 2）结合支架蛋白， VEGF信号传导和缺血后血管生成。然而，ATP 7A在缺血后缺陷性内皮细胞中的作用，糖尿病中的血管重建是完全未知的。初步数据表明，ATP 7A阻止VEGFR 2 并通过调节VE-钙粘蛋白（VE-钙粘蛋白）维持基础EC屏障功能。 Cad）。糖尿病小鼠内皮细胞及2型糖尿病微血管中ATP 7A表达明显降低患者具有降低的Cu转运功能的ATP 7A突变体（ATP 7Amut）小鼠或糖尿病小鼠表现出受损的Cu转运功能。局部缺血诱导的修复性血管生成与过量的组织Cu和血管渗透性/组织损伤，通过过表达ATP 7A来拯救。因此，我们假设ATP 7A的功能是促进和整合关键血管修复程序，如血管生成和维持内皮屏障以Cu依赖性方式发挥作用，这是恢复糖尿病患者新血管形成所必需的。缺血性血管疾病目的1将定义ATP 7A针对以下的保护作用：i）受损的VEGF诱导的细胞凋亡。 ii）通过稳定VEGFR 2来促进血管生成，和ii）通过维持Cu稳态来促进内皮屏障功能障碍。从糖尿病小鼠和2型糖尿病患者的人微血管中分离的EC。目标2将决定糖尿病中ATP 7A下调损害VEGFR 2信号传导和内皮细胞的分子机制 i）ATP 7A与IQGAP 1结合，阻止VEGFR 2泛素化/降解和ii）ATP 7A在调节miR-125 b中的作用，miR-125 b通过Cu-2抑制VE-Cad。依赖性转录因子Atox 1。目的3将确定ATP 7A对糖尿病诱导的糖尿病的保护作用。受损的缺血后新血管形成和体内组织修复，并使用严重肢体缺血的动物模型。我们将使用ATP 7Amut和ATP 7A转基因小鼠;诱导型EC特异性 ATP 7A-/-或Cu导入剂CTR 1-/-小鼠或1型和2型糖尿病小鼠;创新的ICP-质谱，X射线荧光显微镜分析细胞和组织中的[Cu]i;基于FRET或BiFC的蛋白质-蛋白质相互作用; 和活体显微镜检查。我们的研究将揭示铜转运蛋白ATP 7A作为一个新的潜在的治疗靶点加强糖尿病血管并发症患者的综合血管修复计划。